Proteins are essential to all biological functions, from metabolism, to growth, to reproduction, to immunity. As such, they have an important potential role as pharmaceutical agents for the treatment of a wide range of human diseases. Indeed, they have already been used to treat diseases such as cancer, hemophilia, anemia and diabetes successfully, and for a number of diseases are the only effective treatment.
Although protein drugs have enormous therapeutic potential, their more widespread use has been limited by several restrictive technical factors. First, proteins remain difficult and expensive to manufacture compared to other pharmaceuticals. Large-scale purification of proteins in bioactive form can be a limiting step in the commercialization of these drug. Second, many proteins are metabolized or otherwise eliminated quickly in the patient. This results in the need for frequent re-administration. Finally, protein drugs generally must be given by injection. This increases the complexity and expense of the treatment, and the disagreeable nature of administration also limits potential clinical applications.
Delivery of therapeutic gene products (such as polypeptides for protein replacement therapy) by expression in cells transformed with a therapeutic gene product-encoding DNA has attracted wide attention as a method to treat various mammalian diseases and enhance production of specific proteins or other cellular products. This promising technology, often referred to as gene therapy, is generally accomplished by introducing exogenous genetic material into a mammalian patient's cells. The introduced genetic material can be designed to replace an abnormal (defective) gene of the mammalian patient ("gene replacement therapy"), or can be designed for expression of the encoded protein or other therapeutic product without replacement of any defective gene ("gene augmentation"). Because many congenital and acquired medical disorders result from inadequate production of various gene products, gene therapy provides a means to treat these diseases through either transient or stable expression of exogenous nucleic acid encoding the therapeutic product.
Delivery of therapeutic gene products by expression in transformed cells can be accomplished by either direct transformation of target cells within the mammalian subject (in vivo gene therapy) or transformation of cells in vitro and subsequent implantation of the transformed cells into the mammalian subject (ex vivo gene therapy). A variety of methods have been developed to accomplish in vivo transformation including mechanical means (e.g., direct injection of nucleic acid into target cells or particle bombardment), recombinant viruses, liposomes, and receptor-mediated endocytosis (RME) (for reviews, see Chang et al. 1994 Gastroenterol. 106: 1076-84; Morsey et al. 1993 JAMA 270: 2338-45; and Ledley 1992 J. Pediatr. Gastroenterol. Nutr. 14: 328-37).
As with all therapies, the therapy that is most easily administered, least expensive, and most likely to realize patient compliance is the therapy of choice. Intestinal gene therapy provides such a therapy in the realm of gene therapy techniques. The intestinal epithelium is a particularly attractive site for in vivo gene therapy, largely due to the ease of access via an oral or other lumenal route, thus allowing administration of the exogenous nucleic acid via non-invasive procedures. For example, the patient can simply take a pill composed of the exogenous nucleic acid or alternatively the exogenous nucleic acid formation can be administered by some other non-invasive means (i.e., a means that does not require a major surgical procedure, such as endoscopic catheterization or rectal suppository incision).
However, past efforts to accomplish in vivo transformation of intestinal cells have met with severe obstacles. Because the field has been primarily concerned with long-term transformation and delivery of the therapeutic gene product of interest, most groups have shunned intestinal epithelial cells as targets for transformation due to the cells' rapid turn-over rate (2 to 4 days) (see, e.g., Sandberg et al. 1994 Hum. Gene Therap. 5: 303-9). Efforts to achieve in vivo transformation may be further complicated by the mucus layer of the intestine, which is thought to block access of the gene therapy transforming formulation to the target cells (Sandberg et al., supra). The presence of high concentrations of DNAses in the intestinal tract is also thought to be a formidable barrier to the effective introduction of DNA into intestinal tract cells.
Many of the vectors and delivery systems developed for in vivo cellular transformation either have their own inherent drawbacks or are not entirely suitable for in vivo intestinal cell transformations. For example, recombinant viruses, particularly retroviruses, may be slow in gaining FDA approval due to concerns generally associated with the administration of live viruses to humans. In addition, it has become clear that viral vectors present problems with the possibility of multiple administrations of the gene construct due to immune responses, and may greatly limit their utility. Mechanical means, such as the gene gun, are designed for use in transformation of skeletal muscle cells and are not particularly useful in intestinal cell transformation due to problems of access and to the delicate nature of organ.
Current methods for drug delivery by transformation that are designed to accomplish systemic therapeutic goals (e.g., to accomplish administration of protein-based drugs) include both ex vivo and in vivo techniques. However ex vivo techniques require complex procedures to accomplish transformation, put the subject at risk of rejection of the transplant, require at least minor invasive procedures, and limit implantation to modest numbers of cells. In vivo methods (e.g., direct administration through blood or to muscle) also frequently require invasive procedures and meet with difficulties in delivery of the transforming material to the target cell. Moreover, delivery of the transforming material via the bloodstream of the individual results in exposure of the DNA and any carrier associated with it to the immune system, which can result in adverse reactions (e.g., inflammatory reactions to the DNA administered and/or to components of the formulation containing the DNA.
Today, as the biomedical research enterprise discovers new proteins at an increasing pace, and as known proteins become available as therapeutic agents, there is a vital need to develop new delivery systems and methods to expand the application of these molecules as drugs by improving the feasibility and convenience of their use. The present invention addresses these problems.